Prokaryotic Expression And Polyclonal Antibody Preparation Of F3H, A Key Enzyme In The Secondary Metabolism Of Tartary Buckwheat

Fagopyrum tataricum (buckwheat), as an important member of the Polygonaceae family,is rich in flavonoids and mainly produced in high altitude area. It is well known thatflavonoids have significant medicinal value of preventing and treating diseases, such asdiabetes, high cholesterol and high blood pressure. Flavonoids-3-hydroxylase (F3H) is a keyenzyme in the metabolic pathway of flavonoids, belonging to2-ketoglutarate-dependentdioxygenase family. With the help of the cofactors:2-oxoglutarate, molecular oxygen, ferrumand ascorbic acid, F3H catalyzes the conversion of naringenin to dihydrokaempferol (DHK),which is the precursor of flavonol, flower pigments, anthocyanins and other substances. Tothe best of our knowledge, studies of F3H were mainly concentrated in ornamental plants.However, little is know in buckwheat. In this study, we achieved truncatedFlavanone-3-hydroxylase gene (TrF3H) from cDNA library of tartary buckwheat in theseed-filling period. The following researches have been conducted:Sequence and structure of F3H gene were analyzed using bioinformatic approaches. Theresults revealed that F3H was1369bp in length and encoded367amino acids. Phylogeneticanalysis revealed that Fagopyrum tataricum F3H amino acid had high similarity toFagopyrum esculentum (83%). Similarly, the phylogenetic tree also indicated that it has aclose relationship with Fagopyrum esculentum. Structurally, F3H protein consisted of threemajor domain, a highly conserved2OG-FeII-Oxy domain, PLN03176and PLN02515domains. In current study, we cloned639bp truncated F3H gene encoding213amino acids atthe c-terminal of coding sequence, the molecular weight and pI were24.81kD and6.36,respectively.Based on the recombinant plasmid pTriplEx2-TrF3H obtained from full-length cDNAlibrary of Tartary buckwheat, we amplified the truncated coding region of F3H (TrF3H).Prokaryotic expression vector pET47b-TrF3H was constructed and transformed into E. coliRosetta plysS (DE3), after that the expression of TrF3H was indentified by SDS-PAGE. Theresults indicated that TrF3H was highly expressed in the form of inclusion bodies.Overexpressed target protein was detected by gelcode His-tag staining after beingpurified by cobalt chelating chromatography. SDS-PAGE result demonstrated that the singleband could be seen in gel stained with CBB R250and Gelcode His-tag Stain. When the target fragment was purified by cobalt chelating chromatography andSDS-PAGE, the high titer polyclonal antiserum raised against rabbit was obtained usingTrF3H. Western blotting analysis showed the raised antibody could specifically react with theantigen while native F3H existed in immature seed protein. These results laid the basis forfurther exploration of the functions of F3H in Tartary buckwheat.